The present invention relates to methods and compositions for treating selected conditions of the central and peripheral nervous systems employing non-synaptic mechanisms. More specifically, one aspect of the present invention relates to methods and materials for treating seizures and seizure disorders, epilepsy, status epilepticus, migraine headache, cortical spreading depression, intracranial hypertension, neuropsychiatric disorders, central nervous system edema; for treating or protecting from the pathophysiological effects of toxic agents such as ethanol and certain infectious agents; for treating the pathophysiological effects of head trauma, stroke, ischemia and hypoxia; and for improving certain brain functions, such as cognition, learning and memory by administering agents that modulate ionic concentrations and ionic balances in the central nervous system. Specific treatment compositions, including loop diuretics, analogs and derivatives of such compositions, as well as combinations of such compositions with other agents for modulating ionic concentrations and gradients, and for treating various conditions, are disclosed. Materials and methods for treating pain by administering agents that modulate ionic concentrations and gradients in the peripheral nervous system are also disclosed. Methods and systems for screening drug candidate compounds for desired activities using in vitro and in vivo systems are described.
Conventional treatments for neuronal disorders, such as seizure disorders, epilepsy, and the like, target synaptic mechanisms that affect excitatory pathways, such as by modulating the release or activity of neurotransmitters or inhibitors. Conventional treatment agents and regimen for seizure disorders diminish neuronal excitability and inhibit synaptic firing. One serious drawback of this approach is that while seizures are generally localized, the treatment affects (diminishes) neuronal activity indiscriminately. For this reason, there are serious side effects and repeated use of conventional medications may result in unintended deficiencies in normal and desirable brain functions, such as cognition, learning and memory. More detailed information concerning particular disorders of interest is provided below.
Epilepsy
Epilepsy is characterized by abnormal discharges of cerebral neurons and typically manifested as various types of seizures. Epileptiform activity is identified with spontaneously occurring synchronized discharges of neuronal populations that can be measured using electrophysiological techniques. This synchronized activity, which distinguishes epileptiform from non-epileptiform activity, is referred to as xe2x80x9chypersynchronizationxe2x80x9d because it describes the state in which individual neurons become increasingly likely to discharge in a time-locked manner with one another.
Epilepsy is one of the most common neurological disorders, affecting about 1% of the population. There are various forms of epilepsy, including idiopathic, symptomatic and cryptogenic. Genetic predisposition is thought to be the predominant etiologic factor in idiopathic epilepsy. Symptomatic epilepsy usually develops as a result of a structural abnormality in the brain.
Status epilepticus is a particularly severe form of seizure, which is manifested as multiple seizures that persist for a significant length of time, or serial seizures without any recovery of consciousness between seizures. The overall mortality rate among adults with status epilepticus is approximately 20 percent. Patients who have a first episode are at substantial risk for future episodes and the development of chronic epilepsy. The frequency of status epilepticus in the United States is approximately 150,000 cases per year, and roughly 55,000 deaths are associated with status epilepticus annually. Acute processes that are associated with status epilepticus include intractable epilepsy, metabolic disturbances (e.g. electrolyte abnormalities, renal failure and sepsis), central nervous system infection (meningitis or encephalitis), stroke, degenerative diseases, head trauma, drug toxicity and hypoxia. The fundamental pathophysiology of status epilepticus involves a failure of mechanisms that normally abort an isolated seizure. This failure can arise from abnormally persistent, excessive excitation or ineffective recruitment of inhibition. Studies have shown that excessive activation of excitatory amino acid receptors can cause prolonged seizures and suggest that excitatory amino acids may play a causative role. Status epilepticus can also be caused by penicillin and related compounds that antagonize the effects of xcex3-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the brain.
One early diagnostic procedure for epilepsy involved the oral administration of large quantities of water together with injections of vasopressin to prevent the accompanying diuresis. This treatment was found to induce seizures in epileptic patients, but rarely in non-epileptic individuals (Garland et al., Lancet, 2:566, 1943). Status epilepticus can be blocked in kainic acid-treated rats by intravenous injection of mannitol (Baran et al., Neuroscience, 21:679, 1987). This effect is similar to that achieved by intravenous injection of urea in human patients (Carter, Epilepsia, 3:198, 1962). The treatment in each of these cases increases the osmolarity of the blood and extracellular fluid, resulting in water efflux from the cells and an increase in extracellular space in the brain. Acetazolamide (ACZ), another diuretic with a different mechanism of action (inhibition of carbonic anhydrase), has been studied experimentally as an anticonvulsant (White et al., Advance Neurol., 44:695, 1986; and Guillaume et al., Epilepsia, 32:10, 1991) and used clinically on a limited basis (Tanimukai et al., Biochem. Pharm., 14:961, 1965; and Forsythe et al., Develop. Med. Child Neurol., 23:761, 1981). Although its mechanism of anticonvulsant action has not been determined, ACZ does have a clear effect on the cerebral extracellular space.
Traditional anti-epileptic drugs exert their principal effect through one of three mechanisms: (a) inhibition of repetitive, high-frequency neuronal firing by blocking voltage-dependent sodium channels; (b) potentiation of xcex3-aminobutyric acid (GABA)-mediated postsynaptic inhibition; and (c) blockade of T-type calcium channels. Phenytoin and carbamazepine are examples of sodium channel antagonists, which exert their effect at the cellular level by reducing or eliminating sustained high-frequency neuronal depolarization while not appreciably affecting regular firing rates of neurons. Barbiturates, such as Phenobarbital and benzodiazepines, act by enhancing GABA-mediated synaptic inhibition. Both classes of compounds increase the hyperpolarization of the postsynaptic membrane, resulting in increased inhibition. Ethosuximide and valporate are examples of drugs that decrease calcium entry into neurons through T-type voltage-dependent calcium channels.
Current anti-epileptic drug therapies exert their pharmacological effects on all brain cells, regardless of their involvement in seizure activity. Common side effects are over-sedation, dizziness, loss of memory and liver damage. Additionally, 20-30% of epilepsy patients are refractory to current therapy.
Focus on synaptic hyperexcitability has been a guiding principle in basic research on the mechanisms of epileptogenesis and in the design and discovery of new anti-epileptic drugs. One of the shortcomings of this approach is that most current anti-epilepsy drugs exert their influence in an indiscriminate manner, in both the epileptogenic and normal areas in the brain. The compositions of the present invention offer a novel approach to the treatment of seizures, in part because they act via a non-synaptic pathway.
Migraine
Migraine headaches afflict 10-20% of the U.S. population, with an estimated loss of 64 million workdays annually. Migraine headache is characterized by pulsating head pain that is episodic, unilateral or bilateral, lasting from 4 to 72 hours and often associated with nausea, vomiting and hypersensitivity to light and/or sound. When accompanied by premonitory symptoms, such as visual, sensory, speech or motor symptoms, the headache is referred to as xe2x80x9cmigraine with aura,xe2x80x9d formerly known as classic migraine. When not accompanied by such symptoms, the headache is referred to as xe2x80x9cmigraine without aura,xe2x80x9d formerly known as common migraine. Both types evidence a strong genetic component, and both are three times more common in women than men. The precise etiology of migraine has yet to be determined. It is theorized that persons prone to migraine have a reduced threshold for neuronal excitability, possibly due to reduced activity of the inhibitory neurotransmitter xcex3-aminobutyric acid (GABA). GABA normally inhibits the activity of the neurotransmitters serotonin (5-HT) and glutamate, both of which appear to be involved in migraine attacks. The excitatory neurotransmitter glutamate is implicated in an electrical phenomenon called cortical spreading depression, which can initiate a migraine attack, while serotonin is implicated in vascular changes that occur as the migraine progresses.
Cortical spreading depression (CSD) is characterized by a short burst of intense depolarization in the occipital cortex, followed by a wave of neuronal silence and diminished evoked potentials that advance anteriorly across the surface of the cerebral cortex. Enhanced excitability of the occipital-cortex neurons has been proposed as the basis for CSD. The visual cortex may have a lower threshold for excitability and therefore is most prone to CSD. Mitochondrial disorders, magnesium deficiency and abnormality of presynaptic calcium channels may be responsible for neuronal hyperexcitability (Welch, K. M. A., Pathogenesis of Migraine, Seminars in Neurobiology, vol. 17:4, 1997). During a spreading depression event, profound ionic perturbations occur, which include interstitial acidification, extracellular potassium accumulation and redistribution of sodium and chloride ions to intracellular compartments. In addition, prolonged glial swelling occurs as a homeostatic response to altered ionic extracellular fluid composition and interstitial neurotransmitter and fatty acid accumulation. Studies have shown that furosemide inhibits regenerative cortical spreading depression in anaesthetized cats (Read, S J, et al, Cephalagia, 17:826, 1997).
A study of eighty-five patients with refractory transformed migraine type of chronic daily headache (CDH) concluded that acute headache exacerbations responded to specific anti-migraine agents such as ergotamine, dihydroergotamine (DHE), and sumatriptan, and addition of agents such as acetazolamide and furosemide, after diagnosis of increased intracranial pressure, resulted in better control of symptoms (Mathew, N. T. et al. Neurology 46:(5), 1226-1230, May 1996). The authors note that these results suggest a link between migraine and idiopathic intracranial hypertension that needs further research.
Drug therapy is tailored to the severity and frequency of migraine headaches. For occasional attacks, abortive treatment may be indicated, but for attacks occurring two or more times per month, or when attacks greatly impact the patient""s daily life, prophylactic therapy may be indicated. Serotonin receptor agonists, such as sumatriptan, have been prescribed for abortive therapy. Serotonin receptor agonists are thought to constrict dilated arteries of the brain and thereby alleviate the associated pain. Side effects associated with this therapy include tingling, dizziness, warm-hot sensation, land injection-site reactions. Intravenous administration is contraindicated as a consequence of the potential for coronary vasopasms. Ergotamine-based drugs are classified as vasoconstrictors that specifically counteract the dilation of some arteries and arterioles, primarily the branches of the external carotid artery. To prevent ergotamine rebound phenomena, ergotamine should not be repeated on the second or third day of a migraine attack. Yet, if the drug is stopped abruptly, the patient will experience a severe rebound headache. Excessive consumption of ergotamines may cause symptoms of vasoconstriction, such as cold clammy extremities, and may lead to ergotism.
Drugs used for prophylactic indications include andrenergic beta-blockers such as propranolol, calcium channel blockers, or low-dose anti-epileptics. In particular, anti-epileptic drugs that increase brain levels of GABA, either by increasing GABA synthesis or reducing its breakdown, appear to be effective in preventing migraine in certain individuals. In some patients, tricyclic analgesics, such as amitriptline, can be effective. NMDA receptor antagonist, acting at one of the glutamate receptor subtypes in the brain, inhibits CSD. Drugs or substances currently believed to function as weak NMDA receptor antagonists include dextromethoraphan, magnesium and ketamine. Intravenous magnesium has been successfully used to abort migraine attacks.
Neurotoxicity
A variety of chemical and biological agents, as well as some infectious agents, have neurotoxic effects. A common example is the pathophysiological effect of acute ethanol ingestion. Episodic ethanol intoxications and withdrawals characteristic of binge alcoholism result in brain damage. Animal models designed to mimic the effects of alcohol in the human have demonstrated that a single dose of ethanol given for 5-10 successive days results in neurodegeneration in the entorhinal cortex, dentate gyrus and olfactory bulbs, accompanied by cerebrocortical edema and electrolyte (Na+ and, K+) accumulation. As with other neurodegenerative conditions, research has focused primarily on synaptically based excitotoxic events involving excessive glutamatergic activity, increased intracellular calcium and decreased xcex3-aminobutyric acid. Co-treatment of brain damage induced by episodic alcohol exposure with an NMDA receptor antagonist, Non-NMDA receptor and Ca2+ channel antagonists with furosemide reduces alcohol-dependent cerebrocortical damage by 75-85%, while preventing brain hydration and electrolyte elevations (Collins, M., et al, FASEB, vol.12 February 1998). The authors observed that the results suggest that furosemide and related agents might be useful as neuroprotective agents in alcohol abuse.
Cognition, Learning and Memory
The cognitive abilities of mammals are thought to be dependent on cortical processing. It has generally been accepted that the most relevant parameters for describing and understanding cortical function are the spatio-temporal patterns of activity. In particular, long-term potentiation and long-term depression have been implicated in memory and learning and may play a role in cognition. Oscillatory and synchronized activities in the brains of mammals have been correlated with distinct behavioral states.
Synchronization of spontaneous neuronal firing activity is thought to be an important feature of a number of normal and pathophysiological processes in the central nervous system. Examples include synchronized oscillations of population activity such as gamma rhythms in the neocortex, which are thought to be involved in cognition (Singer and Gray, 1995), and theta rhythm in hippocampus, which is thought to play roles in spatial memory and in the induction of synaptic plasticity (Heurta and Lisman 1995; Heurta and Lisman 1996; O""keefe 1993). To date, most research on the processes underlying the generation and maintenance of spontaneous synchronized activity has focused on synaptic mechanisms. However, there is evidence that nonsynaptic mechanisms may also play important roles in the modulation of synchronization in normal and pathological activities in the central nervous system.
Screening of Candidate Compounds and Evaluating Treatment Efficacy
Drug development programs rely on in vitro screening assays and subsequent testing in appropriate animal models to evaluate drug candidates prior to conducting clinical trials using human subjects. Screening methods currently used are generally difficult to scale up to provide the high throughput screening necessary to test the numerous candidate compounds generated by traditional and computational means. Moreover, studies involving cell culture systems and animal model responses frequently don""t accurately predict the responses and side effects observed during human clinical trials.
Conventional methods for assessing the effects of various agents or physiological activities on biological materials, in both in vitro and in vivo systems, generally are not highly sensitive or informative. For example, assessment of the effect of a physiological agent, such as a drug, on a population of cells or tissue grown in culture, conventionally provides information relating to the effect of the agent on the cell or tissue population only at specific points in time. Additionally, current assessment techniques generally provide information relating to a single or a small number of parameters. Candidate agents are systematically tested for cytotoxicity, which may be determined as a function of concentration. A population of cells is treated and, at one or several time points following treatment, cell survival is measured. Cytotoxicity assays generally do not provide any information relating to the cause(s) or time course of cell death.
Similarly, agents are frequently evaluated based on their physiological effects, for example, on a particular metabolic function or metabolite. An agent is administered to a population of cells or a tissue sample, and the metabolic function or metabolite of interest is assayed to assess the effect of the agent. This type of assay provides useful information, but it does not provide information relating to the mechanism of action, the effect on other metabolites or metabolic functions, the time course of the physiological effect, general cell or tissue health, or the like.
U.S. Pat. Nos. 5,902,732 and 5,976,825 disclose methods for screening drug candidate compounds for anti-epileptic activity using glial cells in culture by osoniotically shocking glial cells, introducing a drug candidate, and assessing whether the drug candidate is capable of abating changes in glial cell swelling. This patent also discloses a method for screening drug candidate compounds for activity to prevent or treat symptoms of Alzheimer""s disease, or to prevent CNS damage resulting from ischemia, by adding a sensitization agent capable of inducing apoptosis and an osmotic stressing agent to CNS cells, adding the drug candidate, and assessing whether the drug candidate is capable of abating cell swelling. A method for determining the viability and health of living cells inside polymeric tissue implants is also disclosed, involving measuring dimensions of living cells inside the polymeric matrix, osmotically shocking the cells, and then assessing changes in cell swelling. Assessment of cell swelling activity is achieved by measuring intrinsic optical signals using an optical detection system.
Selected treatment compositions and methods of the present invention are useful for treating central nervous system conditions, including seizures and seizure disorders, epilepsy, including status epilepticus, migraine headaches, cortical spreading depression, intracranial hypertension, neuropsychiatric disorders, and central nervous system edema. Selected treatment compositions and methods of the present invention are also suitable for treating or protecting from the pathophysiological effects of neurotoxic agents such as ethanol and certain infectious agents, and for treating the pathophysiological effects of head trauma, stroke, ischemia and hypoxia. According to another embodiment, treatment agents and methods of the present invention improve function in certain cortical tissue, such as in cortical centers of cognition, learning and memory. Additionally, treatment agents and methods of the present invention are useful for treating pain by affecting or modulating the conduction of impulses associated with pain in the peripheral nervous system. Treatment compositions and methods of the present invention may be used episodically or prophylactically and are suitable for both human and veterinary applications.
Methods and compositions of the present invention involve treatment of various conditions of the central and peripheral nervous systems by means of non-synaptic mechanisms and, more specifically, by modulating the synchronization of neuronal population activity. According to a preferred embodiment, the synchronization of neuronal population activity is manipulated by modulating anionic concentrations and gradients in the central and/or peripheral nervous systems. Ion dependent cotransporter antagonists are suitable treatment compositions, anion cotransporter antagonists are preferred treatment compositions, and cation-chloride cotransporter antagonists are especially preferred treatment compositions. According to one embodiment, Na+,K+,2Clxe2x88x92 chloride cotransporter antagonists are especially preferred treatment agents for modulating the synchronization of neuronal population activity. Anion cotransporter antagonists are useful for treating conditions such as seizures, epilepsy and status epilepticus, cortical spreading depression and migraine headaches, intracranial hypertension, neuropsychiatric disorders, central nervous system edema, for treating or protecting from the pathophysiological effects of neurotoxic agents such as ethanol and certain infectious agents, and for reducing the perception of pain. Chloride cotransporter agonists are preferred treatment agents, and cation-chloride cotransporter agonists are especially preferred treatment agents for improving function in cortical areas associated with cognition, learning and memory, for example. Methods for screening candidate compounds for ion-dependent cotransporter agonist and antagonist activity, and for efficacy in treating various conditions and disorders are also provided.
Reference to the methods and compositions of the present invention using xe2x80x9cnon-synapticxe2x80x9d mechanisms means that mechanisms associated with neuronal excitability, such as the release or activity of transmitters, or the release or activity of inhibitors, are not affected by methods and agents of the present invention. Similarly, ion channels and receptors are not directly affected by methods and compositions of the present invention. Rather, the methods and treatment agents of the present invention affect the synchronization, or relative synchrony, neuronal population activity. Preferred methods and treatment agents of the present invention modulate the extracellular anionic chloride concentration and/or the ion gradients in the central or peripheral nervous system without substantially affecting neuronal excitability.
One aspect of the present invention relates to treatment agents and methods for modulating the synchronization of neuronal discharges by diminishing or eliminating hypersynchronization of neuronal population activity associated with seizures and other pathophysiologies of the central nervous system. In one embodiment, the treatment composition is capable of modulating the anion concentration, preferably the chloride concentration, in the extracellular space in the central nervous system. In a preferred embodiment, the treatment agent is a chloride cotransporter antagonist, and in another preferred embodiment, the treatment agent is a cation chloride cotransporter antagonist, and in an especially preferred embodiment, the treatment composition is a glial cell Na+,K+,2Clxe2x88x92 cotransporter antagonist. According to yet another preferred embodiment, the treatment agent has a high level of cation-chloride cotransporter antagonist activity,in glial cells, and has a lower level of ion-dependent cotransporter activity in neuronal and kidney cells. Preferred agents for treatment of central nervous system conditions are preferably capable of crossing the blood brain barrier, or are administered using delivery systems that facilitate delivery of agents to the central nervous system.
In general, loop diuretics, such as furosemide, bumetanide, ethacrynic acid, and the like, exhibit ion-dependent cotransporter antagonist activity and are suitable for use as treatment compositions of the present invention. Although such loop diuretics produce the desired modulation of the extracellular anionic chloride concentrations and ionic gradients and, hence, modulation of synchronization of neuronal population activity, they may also produce other, undesired effects. Furosemide, for example, acts as a cation-chloride cotransporter antagonist in both glial and neuronal cells, as well as in the kidney. Especially preferred treatment agents of the present invention, exhibiting ion-dependent cotransporter antagonist activity, exhibit a high degree of activity in glial cell populations, and exhibit a lesser degree of activity in neuronal and renal cell populations.
Methods for treating status epilepticus involve administering an ion-dependent cotransporter antagonist, preferably a cation-chloride cotransporter antagonist, in combination with another treatment agent. Furosemide and other loop diuretics are suitable cation-chloride cotransporter antagonists. Experimental studies have shown that furosemide treatment produces a transient and early increase in synchronization of neuronal population activity, followed by a persistent and complete disruption of the hypersynchronization characteristic of epileptiform activity. Treatment of status epilepticus, according to the present invention, involves administration of an ion-dependent cotransporter antagonist, preferably a cation-chloride cotransporter antagonists, such as furosemide, in combination with another agent, such as a barbiturate, that is capable of treating the symptoms associated with the transient and early increase in synchronization of neuronal population activity observed upon administration of furosemide.
Methods and treatment compositions for treating seizures and seizure disorders, epilepsy, migraine headaches, cortical spreading depression intracranial hypertension, neuropsychiatric disorders, and for treating or protecting from the pathophysiological effects of neurotoxic agents, head trauma, stroke, ischemia and hypoxia involve modulating the synchronization of neuronal population activity, preferably by modulating ion gradients in the central nervous system. Ion-dependent cotransporter antagonists are preferred treatment compositions, and cation-chloride cotransporter antagonists are especially preferred treatment compositions. If the ion-dependent cotransporter antagonist treatment composition has activity, for example, with respect to glial cells, but has lower or substantially no activity with respect to neuronal cells, it is suitable for administration alone. If the ion-dependent cotransporter antagonist treatment composition has activity with respect to neuronal as well as other types of cells, it is preferably administered in combination with another agent, such as conventional anti-epleptic or anti-convulsant agent.
Another aspect of the present invention relates to methods and agents for relieving pain, or the perception of pain, by effecting or modulating propagation of action potentials or conduction of impulses in certain nerve fibers in the peripheral nervous system. More specifically, changes in extracellular ionic concentrations and ionic gradients in cells in the peripheral nervous system, affected by ion-dependent cotransporters, diminishes the perception or sensation of pain. Ion-dependent cotransporter antagonists and, preferably, cation-chloride cotransporter antagonists, delivered to the peripheral nervous system, are preferred treatment compositions for the reduction of pain.
Yet another aspect of the present invention relates to methods and agents for enhancing the function of cognitive, learning and memory centers in the central nervous system. Enhanced synchronization of neuronal population activity improves function in centers associated with cognitive abilities, learning and memory in central nervous system cortex. Treatment compositions and methods of the present invention for enhancing cognitive, learning and memory functions involve modulating the synchronization and timing of neuronal population activity, preferably by enhancing synchronization and coordinating timing. According to one embodiment, enhancement of synchronization is achieved by administering an agent capable of modulating extracellular anionic chloride concentrations and ionic gradients in the brain. Ion-dependent cotransporter agonists are preferred treatment agents, and cation-chloride cotransporter agonists are especially preferred. Methods for screening candidate compounds for ion-dependent cotransporter agonist activity are also provided.
Screening methods and systems of the present invention employ optical, or spectroscopic, detection techniques to assess the physiological state of biological materials including cells, tissues, organs, subcellular components and intact organisms. The biological materials may be of human, animal, or plant origin, or they may be derived from any such materials. Static and dynamic changes in the geometrical structure and/or intrinsic optical properties of the biological materials in response to the administration of a physiological challenge or a test agent, are indicative and predictive of changes in the physiological state or health of the biological material.
Two different classes of dynamic phenomena are observed in viable biological materials using optical detection techniques: (1) geometrical changes in the diameter, volume, conformation, intracellular space of individual cells or extracellular space surrounding individual cells; and (2) changes in one or more intrinsic optical properties of individual cells or of cell populations, such as light scattering, reflection, absorption, refraction, diffraction, birefringence, refractive index, Kerr effect, and the like. Both classes of phenomena may be observed statically or dynamically, with or without the aid of a contrast enhancing agent. Geometrical changes may be assessed directly by measuring (or approximating) the geometrical properties of individual cells, or indirectly by observing changes in the optical properties of cells. Changes in optical properties of individual cells or cell populations may be assessed directly using systems of the present invention.
Observation and interpretation of geometrical and/or intrinsic optical properties of individual cells or cell populations is achieved in both in vitro and in vivo systems without altering characteristics of the sample by applying physiologically invasive materials, such as fixatives. Physiologically non-invasive contrast enhancing agents, such as vital dyes, may be used in desired applications to enhance the sensitivity of optical detection techniques. In applications employing contrast enhancing agents, the optical detection techniques are used to assess extrinsic optical properties of the biological materials.
Detection and analysis of the geometrical and/or intrinsic optical properties of individual cells or sample cell populations provides information permitting classification of the physiological state of individual cells or sample cell populations. Based on analysis of the geometrical and/or optical properties of a sample cell population, the sample may be classified as viable or non-viable, apoptotic, necrotic, proliferating, in a state of activity, inhibition, synchronization, or the like, or in any of a variety of physiological states, all of which produce distinct geometrical and/or optical profiles. The methods and systems of the present invention therefore provide for identification of the physiological state of a sample population and differentiation among various physiological states.
An important application of the methods and systems of the present invention involves screening cell populations to assess the effect(s) of exposure to various types of test agents and test conditions. The effect of various test agents and conditions may be evaluated on both normal and pathological sample populations. Safety and cytoxicity testing is conducted by exposing a sample population to a test agent or test condition and assessing the physiological state of the sample population using optical techniques at one or more time points following administration of the test agent or test condition. Such testing may be conducted on various sample populations to determine how a test agent or condition affects a desired target sample population, as well as to predict whether a test agent or condition produces physiological side effects on sample populations that are not the target of the test agent or condition.
According to a preferred embodiment, a pathological condition is simulated in biological materials prior to administration of a test agent or test condition to assess the suitability of the test agent or condition for treating the disease state or compromised condition. Exposure of sample populations to a physiological challenge, such as a change in extracellular osmolarity or ion concentration, altered oxygen or nutrient or metabolite conditions, drugs or diagnostic or therapeutic agents, a disturbance in ion homeostasis, electrical stimulation, inflammation, infection with various agents, radiation, and the like, may simulate a pathological state at a cellular or tissue level. Subsequent exposure of the sample populations a test agent or condition and detection and analysis of changes in geometrical and/or optical properties of the sample populations provides information relating to the physiological state of the sample populations produced by the test agent or condition. Screening techniques may be adapted for use with various types of cell sample populations maintained in vitro under appropriate cell culture conditions to provide a high throughput, automated screening system. Alternatively, screening techniques may be adapted to examine cell and tissue populations using various animal models to assess the effect of a physiological challenge and/or administration of a test agent on various cell populations in animal models in situ. Moreover, because the screening techniques of the present invention are physiologically non-invasive and use spectroscopic techniques, they may be adapted to examine cell and tissue populations in humans, in situ, to assess or monitor a patient""s condition, and to assess or monitor the efficacy of a treatment agent or regimen.
Changes in geometrical and/or optical properties of individual cells or cell populations may be determined by reference to empirically determined standards for specific cell types, cell densities and various physiological states, or appropriate controls may be run in tandem with the test samples to provide direct comparative data. Data is collected and, preferably, stored at multiple time points to provide data relating to the time course of the effect of a test agent or condition on sample populations. Strategies for designing screening protocols, including appropriate controls, multiple samples for screening various dosages, activities, and the like, are well known in the art and may be adapted for use with the methods and systems of the present invention.